Request for a Temporary Exemption from the Requirement of a 

Tolerance

Aspergillus flavus strains TC16F, TC35C, TC38B and TC46G in or 

on Corn

EPA BIOPESTICIDES AND POLLUTION PREVENTION DIVISION COMPANY 

NOTICE OF FILING FOR PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL 

REGISTER 

EPA has received a pesticide petition from Interregional Research Project Number 4 (IR-4), 

Rutgers University, 500 College Road East, Suite 201W, Princeton, NJ 08540 (on behalf of the 

Texas Corn Producers Board, 4205 North Interstate 27, Lubbock, TX 79403), proposing,

pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 

346a(d), to amend 40 CFR part 180 to establish a temporary tolerance exemption for the 

microbial pesticides Aspergillus flavus strains TC16F, TC35C, TC38B and TC46G in or on corn.

The purpose of this petition is to establish a temporary tolerance exemption associated with an 

experimental use permit.

Pursuant to section 408(d)(2)(A)(i) of the FFDCA, as amended, IR-4 (on behalf of the Texas 

Corn Producers Board) has submitted the following summary of information, data, and 

arguments in support of the pesticide petition. This summary was prepared by IR-4 (on behalf of 

the Texas Corn Producers Board), and EPA has not fully evaluated the merits of the pesticide 

petition. The summary may have been edited by EPA if the terminology used was unclear, the 

summary contained extraneous material, or the summary unintentionally made the reader 

conclude that the findings reflected EPA’s position and not the position of the petitioner. 

I. IR-4 (on behalf of the Texas Corn Producers Board) Petition Summary

 

A. Product Name and Proposed Use Practices 

A. flavus strains TC16F, TC35C, TC38B and TC46G, non-aflatoxin-producing strains of A. 

flavus, are proposed for application to corn to reduce the incidence of aflatoxin-producing strains 

of A. flavus and thereby reduce aflatoxin contamination of corn. A. flavus strains TC16F, TC35C, 

TC38B and TC46G, which do not produce aflatoxin, competitively exclude aflatoxin-producing 

A. flavus strains without increasing A. flavus in the environment in the long term. A sterile carrier 

colonized with A. flavus strains TC16F, TC35C, TC38B and TC46G is applied at 10 pounds of 

end-use product per acre (total amount of active ingredients less than 0.01 lb/acre). The current 

submission proposes to establish a temporary exemption from tolerance for residues of A. flavus

strains TC16F, TC35C, TC38B and TC46G in or on corn. The experimental use permit is for up 

to 20,000 acres of corn in Texas, Oklahoma, Arkansas and Louisiana.
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B. Product Identity/Chemistry 

1. Identity of the pesticide and corresponding residues. The pesticide and corresponding 

residues are identified as A. flavus strains TC16F, TC35C, TC38B and TC46G. All four active 

ingredients are included on a carrier on the end-use product FourSure.

2. Magnitude of residue at the time of harvest and method used to determine the residue. 

A. flavus strains TC16F, TC35C, TC38B and TC46G are naturally occurring fungi that were

originally isolated from cottonseed produced in Texas. A. flavus strains TC16F, TC35C, TC38B 

and TC46G have been shown to be naturally and consistently associated with corn. A. flavus is a 

widespread fungus. It is particularly well adapted to the hot regions of the U.S. where it is 

widespread in the environment. The communities of A. flavus in the desert and in agricultural 

fields are naturally composed of both aflatoxin-producing (toxigenic) and aflatoxin nonproducing (atoxigenic) strains. Both atoxigenic and toxigenic strains have been found on 

essentially all plant material and soils in the southwestern U.S. The goal of applications is to 

increase the percent of the A. flavus community composed of the strains TC16F, TC35C, TC38B 

and TC46G and to decrease the percent of A. flavus that produces aflatoxin on the crop and the 

field.

3. A statement of why an analytical method for detecting and measuring the levels of the 

pesticide residue are not needed. A temporary exemption from the requirement of a tolerance for

residues of the microbial pesticides A. flavus strains TC16F, TC35C, TC38B and TC46G is being 

proposed for corn. All lots of the active ingredients (A. flavus strains TC16F, TC35C, TC38B 

and TC46G) and the formulated product are monitored as part of a rigorous quality control 

program. Starter cultures of A. flavus used in the production of the end-use product are always 

screened for aflatoxin production using thin layer chromatography (TLC) and appropriate 

standards. Quality control standards are zero tolerance in the starter cultures and in the 

formulated product for aflatoxin production and for A. flavus not identified as A. flavus strains

TC16F, TC35C, TC38B or TC46G. A. flavus strains TC16F, TC35C, TC38B and TC46G have

never been found to produce aflatoxin. Starter cultures of A. flavus strains TC16F, TC35C, 

TC38B and TC46G as well as the end-use product containing these active ingredients are also 

identified to strain by vegetative compatibility analysis.

C. Mammalian Toxicological Profile

There are existing registrations on other atoxigenic strains of A. flavus, including AF36 and 

NRRL 21822, and it is reasonable to expect these new native strains would be similar to the 

existing registered products. An acute oral toxicity test was performed whereby a single oral dose 

of 5,000 milligrams/kilogram (mg/kg) per animal of A. flavus strain AF36 colonized carrier was 

administered by gavage to five male and five female Sprague Dawley rats. The oral LD50 was 

determined to be greater than 5,000 mg/kg rat body weight. No clinical signs were observed 

during the 14-day study, and no abnormalities or adverse effects were observed in any of the rats 

upon necropsy.
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An initial pulmonary rat study resulted in lethality in a significant number of animals treated with 

either the live A. flavus strain AF36 with another ingredient or heat killed A. flavus strain AF36 with 

another ingredient. Onset of symptoms was rapid after dosing with all deaths occurring by day four 

of the study. All rats surviving to day four of the study recovered, and all rats sacrificed (as 

scheduled) on day 8 or day 15 of the study had totally eliminated viable A. flavus strain AF36 from 

the lungs, caecal contents, and feces. There was no evidence of infectivity. The aetiology of deaths 

was unclear. It appeared that A. flavus strain AF36 prepared using the other ingredient caused a 

severe acute inflammatory response. Retrospective literature review and consultation with a 

toxicologist supported the theory that the responses were a result of a synergism with the other 

ingredient and/or of the other ingredient’s breakdown products formed during preparation of the 

spore suspension.

A second rat study was therefore undertaken. In the second study, the conidia were both washed from 

the carrier and suspended in a different ingredient than in the first study. Animals (2 male and 2 

female for each treatment level) were dosed at 0, 105

, 106, 107, and 108 colony-forming units per rat. 

There were no clinical signs in any of the treatment groups considered to be associated with the test 

substance. Rats were sacrificed at day 8 without treatment-associated mortality. No abnormalities 

were observed in any of the animals at the macroscopic examination at termination. 

Based on these two mammalian studies, it was concluded that A. flavus strain AF36 does not present 

either a toxicological or an infectious risk to mammals. 

Data waivers were requested for the toxicology studies listed below on the following bases:

(i) Researchers and other workers have worked with A. flavus strains at the Southern Regional 

Research Center for over 10 years, in commercial fields since 1996, and in hand-picked field plots 

(1989 to 1994) without report of any adverse health effects; 

(ii) A. flavus strains TC16F, TC35C, TC38B and TC46G are widely distributed in the environment, 

and their occurrence is natural; and 

(iii) The label will require applicators and other handlers to wear Personal Protective Equipment 

(PPE) such as waterproof gloves, a dust/mist filtering respirator with the appropriate NIOSH 

approval prefix N-95, P-95, or R-95, coveralls, long sleeved shirt and long pants, shoes, socks, and 

goggles; therefore, exposure should not be a problem. Applications will involve aerial or ground 

application by mixers/handlers who are licensed and trained to even handle restricted materials. At 

the 10 lb/acre application rate of the formulated material, the total amount of active ingredients is 

less than 0.01 lb/acre. Applications of A. flavus strains TC16F, TC35C, TC38B and TC46G do not 

significantly impact the total amount of A. flavus in the soil or crop, but only change the proportion 

of the atoxigenic A. flavus in relation to the overall soil or crop population. Since the product is 

applied as a granular formulation on a carrier, exposure from drift is minimal.
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1. Acute dermal toxicity. This study was requested to be waived based upon the lack of toxicity in 

animals dosed orally with A. flavus strain AF36. While A. flavus has been reported to be a dermal 

sensitizer, this testing is not warranted, since the PPE required on the label will provide adequate 

protection from dermal exposure to workers and pesticide handlers.

 

2. Primary dermal irritation. This study was requested to be waived based upon the lack of toxicity 

in animals dosed orally with A. flavus strain AF36. Aerial or ground applications will be made by 

mixers/handlers and applicators who are required to wear appropriate PPE and are licensed and 

trained to even handle restricted materials. At the 10 lb/acre application rate of the formulated 

material, the total amount of active ingredient is less than 0.01 lb/acre. Applications do not 

significantly impact the total amount of A. flavus in the soil or crop, but only change the proportion 

of the atoxigenic strain in relation to the overall population. Since the product is applied as a granular 

formulation on a carrier, exposure from drift is minimal.

3. Primary eye irritation. This study was requested to be waived based upon the use of appropriate 

PPE, including goggles, and other factors listed above. Since the product is applied by aerial or 

ground equipment as a granular formulation on a carrier, exposure from drift is expected to be 

minimal.

4. Acute intraperitoneal. This study was requested to be waived based upon the lack of toxicity in 

animals dosed orally and by intratracheal instillation with A. flavus strain AF36. In addition, the 

factors listed above will mitigate exposure, which is expected to be minimal.

5. Genotoxicity, reproductive and developmental toxicity, subchronic toxicity and chronic toxicity 

testing were not performed, since no adverse effects were anticipated in the acute toxicology Tier 1 

studies. Tier II studies are only required if triggered by adverse effects observed in Tier I studies. 

D. Aggregate Exposure

1. Dietary exposure. 

i. Food. A. flavus strains TC16F, TC35C, TC38B and TC46G are naturally occurring 

organisms that do not produce aflatoxin and are thus safer than A. flavus that produces aflatoxin. 

Proposed uses and application rates will not result in increases in the total population of A. flavus

on the mature crop beyond naturally occurring background levels. A. flavus strains TC16F, 

TC35C, TC38B and TC46G, when applied to the soil just prior to tasseling, have been shown to 

significantly reduce the levels of aflatoxin in corn at harvest without changing the quantity of A. 

flavus on the crop. Furthermore, the proposed uses and application rates will not increase 

exposure of humans to A. flavus by dietary means. Applications of A. flavus strains TC16F, 

TC35C, TC38B and TC46G do not increase the indigenous populations of A. flavus associated 

with the harvested crop. The applications merely alter the composition of the fungal community 

associated with the mature crop, so that aflatoxin-producing strains are far less frequent. The 

result is a much lower incidence of aflatoxin in the crop and in the environment associated with 
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the developing and mature crop.

ii. Drinking water. A. flavus strains TC16F, TC35C, TC38B and TC46G are naturally 

occurring organisms that are already widespread in the environment and are not considered to be 

a risk to drinking water. Both percolation through soil and municipal treatment of drinking water 

would reduce the possibility of exposure of A. flavus through the drinking water. Applications of 

A. flavus strains TC16F, TC35C, TC38B and TC46G do not increase the long-term population of 

A. flavus in the environment, and thus are not expected to influence the relationship of A. flavus

to water sources. Applications merely change the composition of the A. flavus community, so 

that aflatoxin are less common in the environment. 

2. Non-dietary exposure. The potential for non-occupational, non-dietary exposure to the 

general population is not expected to be significant and is not expected to present any risk of 

adverse health effects.

E. Cumulative Exposure 

There are existing registrations on other atoxigenic strains of A. flavus, including AF36 and 

NRRL 21822, and it is reasonable to expect these new native strains would be similar to the 

existing registered products whose sole mode of action is to reduce aflatoxin. There are no other 

registered products containing A. flavus strains TC16F, TC35C, TC38B or TC46G, and no 

reasonable concern that there would be any cumulative effects since the active ingredients do not 

have any toxic endpoints. Data submitted show that the fungal metabolite of concern, which is 

aflatoxin, is not produced by A. flavus strains TC16F, TC35C, TC38B or TC46G in the crop or in

artificial media in the lab. A. flavus strains TC16F, TC35C, TC38B and TC46G have been shown 

to exclude aflatoxin-producing fungi competitively from the developing crop and to reduce 

aflatoxin contamination of corn. Data show that the proposed use will not result in appreciable 

increases in the long-term population of A. flavus on the crop beyond naturally occurring levels. 

Furthermore, there is no expectation of cumulative effects with other pesticides.

F. Safety Determination 

1. U.S. population. A. flavus strains TC16F, TC35C, TC38B and TC46G are naturally 

occurring organisms. These strains have low toxicity as demonstrated by the acute oral toxicity 

study in rats by A. flavus strain AF36. A. flavus is ubiquitous throughout the hot desert valleys in 

Arizona. Studies have shown that treatment of corn fields just prior to tasseling with a sterile 

carrier colonized by A. flavus strain AF36 at 10 pounds per acre does not increase the long-term 

population of A. flavus either on the crop at maturity or in the soil. Based on this information on

A. flavus strain AF36, IR-4 is of the opinion that the aggregate exposure to A. flavus over a 

lifetime should not change with application of A. flavus strains TC16F, TC35C, TC38B and 

TC46G, and exposure to both aflatoxin-producing A. flavus strains and aflatoxin should decrease. 

This should be beneficial to human health. Thus, there is a reasonable certainty that no harm will 

result from aggregate exposure to A. flavus strains TC16F, TC35C, TC38B and TC46G.
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2. Infants and children. Based on natural occurrence and the lack of toxicity, there is 

reasonable certainty that no harm to infants, children, or adults will result from aggregate 

exposure to A. flavus strains TC16F, TC35C, TC38B and TC46G. Exempting A. flavus strains

TC16F, TC35C, TC38B and TC46G from the requirement of a tolerance should pose no 

significant risk to humans.

G. Effects on the Immune and Endocrine Systems

A. flavus strains TC16F, TC35C, TC38B and TC46G are naturally occurring organisms that do 

not produce aflatoxin and are thus safer than A. flavus that produces aflatoxin. To date, there is 

no evidence to suggest that A. flavus strains TC16F, TC35C, TC38B and TC46G function in a 

manner similar to any known hormone or that they act as endocrine disrupters.

H. Existing Tolerances

There is no existing tolerance exemption for A. flavus strains TC16F, TC35C, TC38B and 

TC46G. There are permanent tolerance exemptions for A. flavus strain AF36 on cotton, corn and 

pistachio (40 CFR § 180.1206) and A. flavus strain NRRL 21882 on peanut and corn (40 CFR §

180.1254).

I. Efficacy Data

The following information was presented for a tolerance exemption for A. flavus strain AF36 on 

cotton, corn and pistachio. The information is relevant for A. flavus strains TC16F, TC35C, TC38B 

and TC46G and for this request for a temporary exemption from the requirement of a tolerance in or 

on corn, as part of an experimental use permit. Existence of aflatoxin in the environment is a public 

health hazard; as such, data were submitted to demonstrate that proper use of A. flavus strain AF36 

results in reductions in the average aflatoxin-producing potential of fungi resident in treated areas 

and in reductions in the quantity of aflatoxin in crops. In field tests with A. flavus strain AF36, the 

aflatoxin content of cottonseed was shown to be inversely related to the proportion of the A. flavus

community on the crop composed of A. flavus strain AF36. Detailed analyses of the aflatoxin content 

of commercial fields from 1996 through 1998 confirmed that reduced aflatoxin levels were 

associated with displacement of aflatoxin producers from treated crops and that treatments were 

associated with up to 90% reductions in crop aflatoxin content. Efficacy of applications of A. flavus 

strain AF36 was demonstrated for fungal communities both on cottonseed and corn from treated 

crops at harvest and in soils of treated fields one year after treatment. This included cotton crops 

treated in 1996 (112 acres treated), 1997 (463 acres treated), 1998 (499 acrestreated), 1999 (10,488 

acrestreated), 2000 (16,725 acrestreated) and 2001 (19,975 acres treated). Displacement of aflatoxin 

producers is indicated by the proportion of A. flavus communities composed of A. flavus strain 

AF36. In 1996, average incidence of A. flavusstrain AF36 on treated crops was 88.5% and in the soil 

one year after treatment 85.2%. Incidences of A. flavus strain AF36 on treated crops were 78% and 

67% in 1997 and 1998, respectively, and in the soil one year after treatment incidences were 72% 
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and 77%, respectively. Successful displacement was also observed as the acreage treated rapidly 

expanded from 1999 to 2001 with average incidences of A. flavus strain AF36 on treated crops 

ranging from 57% in 1999 to 66% in 2001. Applications were particularly effective at displacing the 

high aflatoxin-producing S strain of A. flavus. S strain isolates produce more aflatoxin than other A. 

flavus and are prominent in soils of cotton-producing areas of California, Arizona and South Texas. 

There are preliminary data indicating that A. flavus strains TC16F, TC35C, TC38B and TC46G

(included on a carrier on the end-use product FourSure) are effective in corn as well. Efficacies 

of the four atoxigenic A. flavus strains selected as active ingredients were assessed in laboratory 

studies on mature kernels. This type of assay reflects increases in toxin that occur in the field 

when the mature crop receives a rain or when both high humidity and high temperature exist after 

maturation. This type of assay also reflects efficacy that is relevant after the crop has been 

harvested, and maize with moisture content above 15% is transported, stored, or even mixed into 

feed and fed (i.e., in the yard at a dairy). All four atoxigenic strains were highly effective at 

reducing contamination in these assays.

All four atoxigenic A. flavus strains on a carrier in the end-use product FourSure demonstrated 

excellent ability to displace aflatoxin producers and modify the fungal community associated with 

crops in a field trial. In three tests over two years (2013 and 2014), A. flavusstrains TC16F, TC35C, 

TC38B and TC46G dispersed to the crop and successfully competed with aflatoxin producers. Thus, 

the modified A. flavus community was more diverse than when a single atoxigenic genotype is 

applied as with A. flavus strain AF36 and A. flavus strain NRRL 21882. There were differences 

among years, locations, and replicates in which strain was the most successful. This suggests that 

slight differences in the crop or environment caused changes in the most successful genotype and 

further supports use of multiple atoxigenic A. flavus strains to provide the best efficacy across the 

varying crop environments that occur between seasons, among locations, and even within individual 

fields. 

The purpose of this temporary exemption from tolerance is to cover the use of A. flavus strains 

TC16F, TC35C, TC38B and TC46G under an experimental use permit. 

J. International Tolerances

There are no Codex maximum residue levels established for residues of A. flavus strains TC16F, 

TC35C, TC38B and TC46G. A. flavus strains TC16F, TC35C, TC38B and TC46G-containing 

products are presently not registered for pest control outside of the United States.